Project Summary/Abstract The NF-?B family of transcription factors regulates diverse biological processes, including immune and inflammatory responses. NF-?B activation involves two major pathways: the canonical and noncanonical pathways. A central step in the noncanonical pathway is ubiquitin-dependent processing of the NF-?B2 precursor protein p100, which generates mature NF-?B2 p52 and cause nuclear translocation of the p100- sequestered noncanonical NF-?B members, p52 and RelB. During the previous funding cycles, the PI's laboratory has made pivotal contributions to the advancement of this relatively new field, including discovery of NIK as a central signaling component and identification of TRAF3 as a primary negative regulator that controls the fate of NIK. It is now known that TRAF3 functions as the substrate binding subunit of a NIK-specific E3 ubiquitin ligase complex, TRAF3-TRAF2-cIAP. Despite these progresses, our knowledge on noncanonical NF- ?B signaling is still quite limited. In particular, only few regulators of this pathway have been characterized, and the function of noncanonical NF-?B in different types of immune cells is poorly defined. The overall objective of this continuation application is to characterize novel regulators and functions of the noncanonical NF-?B pathway. The proposed studies are based on innovative preliminary data from the PI's laboratory. Using newly generated NIK-conditional KO mice, we demonstrated a dendritic cell (DC)- specific function of noncanonical NF-?B in regulating mucosal immunity and obtained important clues to the underlying mechanism. To identify novel regulators of the noncanonical NF-?B pathway, we employed a recently developed approach, BioID, to screen for proteins interacting with major components of this pathway. After extensive characterizations, we have identified a TRAF3-binding protein, TFG, and a p100-binding protein, Otub1, which are critically involved in noncanonical NF-?B regulation. TFG is crucial for preventing abnormal NIK accumulation, whereas Otub1 is a deubiquitinase (DUB) that inhibits p100 ubiquitination and processing. Our gene knockdown and KO mouse studies suggest important functions of TFG and Otub1 in immune regulation. These innovative findings form a solid foundation for this continuation application. To accomplish our overall objective, we will (1) elucidate the mechanism by which noncanonical NF-?B functions in DCs to regulate mucosal immunity; (2) characterize novel factors that regulate noncanonical NF-?B signaling; and (3) investigate the immunoregulatory functions of novel noncanonical NF-?B regulators. The PI's laboratory pioneered the discovery of noncanonical NF-?B pathway and has been in a leading position in this area. During the previous funding cycles, we have made seminal discoveries that have been instrumental for the advancement of the filed. We believe that the proposed studies will once again lead to high-impact findings that substantially advance the field. With our extensive experience and the innovative preliminary data, we are in a unique position to carry out the proposed studies.